With the improvement of living conditions, consumers' desire for healthy and green products from natural origin has increased and thus has accelerated the launch activity for products of natural origin. In contrast to chemical synthesis, biological synthesis has the advantages of mild reaction condition, fewer byproducts, lesser environmental pollution, selectivity and simple downstream processing. Hence research directed towards the microbial biosynthetic pathway to produce natural flavor, food stabilizer etc is gaining significance. Natural aromatic compounds including benzaldehyde, vanillin, p-hydroxy benzoic acid are already in demand and represent a very large market size in the flavor and food industry.
Aromatic, aliphatic and alicyclic aldehydes and alcohol are useful intermediates in the chemical, pharmaceutical, food and flavor industries. Chemical methods for conversion of carboxylic acids to aldehyde or alcohol are limited and they usually require prior derivatization (esterification) and product de-blocking with reactants containing competing functional group (JP2010227893A).
An efficient route to synthesize aryl aldehydes and alcohols from aryl acids is to use the reductive enzyme system of white rot Basidiomycetes as biocatalyst. The white rot fungi with reducing activities include Trametes versicolor (Farmer et al., Biochim. Biophys. Acta. 35 (1959) 202-211), Sporotrichum pulverulentum (Ander et al. Arch Microbiol 125 (1980) 189-202), Phlebia radiata (Lundell et al. Appl. Environ. Microbiol. 56 (1990) 2623-2629), Phanerochaete chrysosporium (Muheim et al., Eur. J Biochem. 195 (1991) 369-375), Bjerkandera sp (De Jong et al. Appl. Environ. Microbiol. 60 (1994) 271-277).
U.S. Pat. No. 6,261,814 discloses use of isolated and purified enzyme system of carboxylic acid reductase from Nocardia sp strain, NRRL 5646 as a biocatalyst for the reduction of carboxylic acids. It requires external addition of expensive cofactors like ATP, NADPH which are difficult to regenerate in the system and hinders the isolation of the product. This process is therefore not commercially viable in terms of yield and cost.
Another U.S. Pat. No. 5,866,380 discloses a process for conversion of vanillic acid to vanillin where the white rot fungus is used and it gives only 31% yield with aldehyde. It also discloses the use of adsorbent resin in the fermentation medium.
U.S. Pat. No. 6,162,637 discloses a process for conversion of vanillic acid to vanillin using Phanerochaete chrysosporium, wherein 0.3 g/L substrate concentration is used for 3 days along with 0.3 g/L every day, 10% amberlite XAD resin added to finally yield 0.628 g/L vanillin.
U.S. Pat. No. 6,162,637 discloses uses of Phanerochaete chrysosporium, MIC 247 to reduce vanillic acid to vanillin, wherein 1.8 g/L of vanillic acid is added in doses of 0.3 g/L each day till 6 days which is a very long time for biotransformation. At the end of 6 days, 0.628 g/L of vanillin is accumulated, but only 46.5% conversion of substrate is observed which ultimately contributes to lower productivity of the overall process.
U.S. Pat. No. 6,844,019 discloses use of the organism Micromucor isabellinus (Zyl 849) to convert vanillic acid to vanillin, wherein vanillic acid is used for the growth of the starter culture, resulting in lower biotransformation yield. Overall 18 g of vanillic acid is added and 8.5 g of vanillin is obtained with 43.8% substrate conversion.
Hage and Schoemaker (1999) employed the basidiomycete strain Bjerkandera sp strain B0S55 to reduce p-anisic acid to corresponding aldehyde and alcohol. However total molar yields of aldehyde and alcohol together was 75% only (Hage et al. Appl Microbiol Biotechnol (1999) 52: 834-838). Other substrates studied were veratric acid, 3-chloro-4-methoxybenzoic acid, 3,5-dichloro-4-methoxy benzoic acid, 3,4-dichloro benzoic acid, 4-fluorobenzoic acid and 3-nitrobenzoic acids. All these acids were reduced, however, the fungi established equilibrium between aldehyde and alcohol production. The total molar yield of aldehyde and alcohol was 74-85%. No selectivity with respect to aldehyde and/or alcohol was obtained. And so a further separation technology is required to isolate the individual metabolites. Also the individual yields of corresponding aldehyde and alcohol is very low.
U.S. Pat. No. 7,462,470 discloses vanillin production from vanillic acid using Pycnoporus cinnabarinus CGMCC 1115. 80% of vanillin yield was obtained over 2 g/L of vanillic acid concentration. But the media composition used for culturing the organism contained a very high amount of nitrogen source and also a higher percentage of initial inoculum which ultimately gave the biomass cells in high concentration. So this high biocatalyst concentration converts/biotransforms a higher substrate concentration.
Biocatalytic processes for the manufacture of small, highly functionalized molecules frequently have limited productivity. A common reason for this is the presence of the reaction products that can cause inhibitory or toxic effects (making poor use of the enzyme) or promote unfavourable equilibria (giving low conversions). In each case, the product needs to be removed as soon as it is formed in order to overcome these constraints and hence increase the productivity of the biocatalytic process. ISPR techniques i.e. in-situ product removal techniques are employed where either an adsorbent resin is added in the biotransformation media to capture the product or a suitable solvent is added which extracts the product. However, when an adsorbent resin is added in the biotransformation media, separation of the resin from the biomass is difficult and further depending upon its binding capacity, the amount of resin to be added is also a limitation in cases where a large amount of resin cannot be added. The use of solvent for product capture has its own limitation of posing toxicity to the cells, thus affecting the biotransformation efficiency. Also, in both the cases, the cells once treated with solvent or added with resin cannot be reused for subsequent biotransformations and so these techniques cannot be used for continuous processes.
Microbial reduction of acids to aldehydes posses a common problem of immediate further reduction to alcohols due to aldehyde toxicity to the cells. Also high concentration of aldehyde causes great product repression in the fermentation which results in the decrease in biotransformation efficiency. The selectivity of the process to obtain aldehyde in high concentration and yield is strongly influenced by the pH of the reaction. Most of the reductions are carried out in the pH range of 4-6.
The invention disclosed in the present invention provides a solution to the above mentioned problem of the prior art. The advantage of this invention is selectivity of aldehyde and/or alcohol at higher yields, low production cost due to cheaper media and recycling of the biomass, high final product concentration, and high productivity due to continuous system, easy downstream processing, clean process and eco friendly product.